Actinobacillus actinomycetemcomitans (Aa) is a Gram-negative, facultative coccobacillus which colonizes the human oral cavity and the upper respiratory tract. Microbial, immunological and clinical studies have implicated Aa in the pathogenesis of both adult and juvenile periodontitis. Aa has also been associated with more serious human infections such as endocarditis and soft tissue abscesses. Although the periodontium is believed to be the source of these infections, little is known about the mechanisms used by Aa to maintain itself within the oral cavity and to infiltrate and disseminate in tissues. Pathogens have developed remarkably complex and diverse strategies of host cell infection and tissue dispersion. The invasion mechanisms used by the intestinal pathogens have been investigated extensively. The processes include multiple steps and the involvement of myriad bacterial genes. The invasion of oral pathogens has not been adequately studied. Our laboratory has provided the only extensive study on the invasion of a periodontopathic microorganism. Aa invasion is a dynamic multistep process which involves cross-talk between the host and bacterium. Adhesion and subsequent entry of Aa is correlated with host cell actin rearrangement, and is likely mediated by transferrin receptor. Exit appears to be dependent on microtubule structure. Aa spread to adjacent cells is mediated through host cell surface protrusions. These studies promoted us to propose a model of the Aa invasion process. In this proposal, we plan to concentrate on how the host cell responds to the invasion of Aa. In order to further our understanding of the critical elements of Aa pathogenesis, we propose to (1) evaluate the role of the transferrin receptor in Aa entry into epithelial cells, (2) establish the role of microtubules in Aa intracellular spread, (3) determine the biogenesis of the Aa-containing vacuole, (4) determine the real-time sequence of events in the invasion process, and (5) investigate transient calcium fluxes in the invasion process. An understanding of the elements that play key roles in Aa pathogenesis should provide insights for the development of rational chemotherapeutic interventions in the treatment of periodontal diseases.